1. Field of the Invention
The present invention relates generally to chemical separation processes and equipment, and more particularly to a method and apparatus for purifying proteins and other high molecular weight compounds from complex media, such as biological media.
The selective separation of high molecular weight compounds from one another is of importance to many chemical, biological, and pharmaceutical processes. In particular, the recombinant production of high value proteins generally requires the isolation of a single valuable protein species from a highly complex mixture. For example, transformed cells may be grown in a suitable culture media, and the desired protein recovered from the growing cells, either by harvesting the cells and disrupting them to release the proteins or by collecting conditioned media into which the desired protein has been secreted. In both cases, it is necessary to recover and purify the desired protein from the cell culture.
Protein purification is most often achieved by chromatographic processes such as ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, and the like. Usually, several separation steps will be performed sequentially in order to recover the desired protein at a required level of purity. Because of the high value of many protein products, particularly recombinant proteins, it is of great interest to provide separation methods and techniques which provide for enhanced purity and recovery from complex mixtures.
Heretofore, electrodialysis techniques have not found widespread use in the separation of proteins and other large molecules. Electrodialysis is a process for transferring ionic species from one solution to another across a membrane under the influence of a direct current electrical potential. Typically, such processes are performed in three-compartment cells defined by alternating anionic and cationic membranes. By passing a product stream between the alternate electrode pairs, and applying an electrical potential having the proper polarity, low molecular weight ionic species will be concentrated in dialysate streams which are directed between membranes which alternate with the product streams.
Electrodialysis techniques have not generally been used for separating one high molecular weight species from another high molecular weight species. While electrodialysis for protein fractionation has been described, such fractionation has been achieved indirectly by desalting a product stream so that certain protein(s) selectively precipitate out to enrich a fraction of remaining protein(s). No separation of proteins across a selective membrane is achieved.
It would therefore be desirable to provide improved electrodialysis methods and apparatus which are suitable for separating large molecular weight species, such as proteins, from one another. It would be particularly desirable if such methods and apparatus could selectively separate proteins into different process streams so that they could be separately collected and further processed. Such methods and apparatus should further be economic and be capable of providing high yields and purities of desired product streams.
2. Description of the Background Art
Electrodialysis for the separation of small ionic molecules is described in McRae, "Electrodialysis," in Kirk-Othmer, Encyclopedia of Chemical Technology, vol. 8, pp. 726-738, John Wiley & Sons, New York, 1986. Electrodialysis for the processing of proteins is described in Reed (1985) Genetic Engineering News vol. 5, no. 9, page 10. The purification of protein-containing whey solutions by electrodialysis is described in the following patents: U.S. Pat. Nos. 5,223,107; 4,441,978; 4,396,477; 4,351,710; 4,332,275; and 4,276,140. Other protein electrodialysis processes are described in WO 92/07818 and WO 91/12726. Isoelectric focusing techniques for separating proteins are described in Wenisch et al. (1992) Electrophoreses 13:668-673, and U.S. Pat. Nos. 5,082,548 and 4,971,670.